Stimulation of RBL-2H3 cells through the high affinity Fc receptor (FcepsilonRI) for immunoglobulin E (IgE) leads to the activation of various tyrosine kinases, with phosphorylation and activation of phospholipase Cgamma, which in turn produces two second messengers, inositol 1,4,5-trisphosphate (IP3) and diacylglycerol (DAG), calcium mobilization and degranulation. To understand calcium dependent degranulation, it is necessary to study the mechanisms for calcium mobilization. It is believed that IP3, produced upon stimulation through FcepsilonRI, causes calcium release from intracellular calcium stores to cytosol and emptying of the stores leads to the calcium influx from the outside of the cell to the cytosol. Therefore, the cytosolic calcium increase involves two components: calcium release from intracellular stores and calcium influx. However, we found that in RBL-2H3 cells, there was no correlation between calcium flux and the generation of IP3 following aggregation of FcepsilonRI via antigen-specific IgE and antigen. In search of an alternative pathway involved in the antigen-stimulated calcium response, we found a novel pathway via sphingosine kinase. The stimulation of RBL-2H3 cells with antigen thus also caused activation of sphingosine-1-phosphate (S1P). S1P is known to release calcium from an IP3-sensitive intracellular pool in an IP3-independent manner. The time course and dose dependence of the Ag-induced calcium signal correlated with those of S1P production. Furthermore, the Ag-induced calcium signal was completely inhibited by preincubation of the cells with a selective inhibitor of sphingosine kinase, DL-threo-dihydrosphingosine (DHS). DHS did not inhibit tyrosine phosphorylation of kinases or production of IP3.